CN116144835A - Method for improving sensitivity of identifying EBV-infected lymphocyte subpopulation and application thereof - Google Patents
Method for improving sensitivity of identifying EBV-infected lymphocyte subpopulation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for improving sensitivity of identifying lymphocyte subpopulations infected by EBV and application thereof, belonging to the technical field of medical detection, and mainly comprising the following steps: s1, extracting peripheral blood mononuclear cells; s2, sorting peripheral blood mononuclear cells to obtain lymphocytes with different subgroup types; s3, re-suspending the obtained lymphocytes with different subgroup types by using a culture medium respectively, uniformly spreading the re-suspended lymphocytes in a cell culture pore plate, respectively stimulating the re-suspended lymphocytes with different subgroup types by using IL-2, and collecting the proliferated lymphocytes with different subgroup types; s4, respectively extracting the DNA of the lymphocytes of different subgroup types, measuring the content of the EB virus DNA, and determining the lymphocyte subgroup infected by the EB virus. The method provided by the invention can improve the detection sensitivity of the content of the EB virus DNA in the lymphocytes of different subgroup types, thereby improving the sensitivity of identifying the lymphocyte subgroup infected by the EB virus.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a method for improving sensitivity of identifying lymphocyte subpopulations infected by EBV and application thereof.
Background
Epstein-Barr Virus (EBV), a member of the family herpesviridae, is a linear DNA Virus, the first Virus demonstrated to be associated with human tumorigenesis, particularly common in humans, a common Virus causing respiratory tract infections, whose specific serum antibodies are detected by more than 95% of adults worldwide, and which persists throughout life, with chronic infections in most humans being asymptomatic.
The detection of EBV active infected lymphocyte has important significance for diagnosis and treatment of EB virus and screening of cell therapeutic targets. However, the detection of EBV DNA in lymphocytes of the prior art can distinguish the cell subsets infected by EBV, but if the patient has a small number of lymphocytes, the amount of nucleic acid extracted cannot meet the criterion for detecting EBV DNA, i.e. the detection sensitivity is limited by the patient's number of lymphocytes.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for improving the sensitivity of identifying EBV-infected lymphocyte subpopulations, which enables detection without being limited to the number of lymphocytes of a patient, thereby improving the sensitivity of identifying EBV-infected lymphocyte subpopulations.
It is a second object of the present invention to provide the use of a method for increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations in determining a therapeutic target for an EBV-infectious disease.
It is a further object of the present invention to provide the use of a method for increasing the sensitivity of identifying EBV-infected lymphocyte subsets for guiding the preparation of a medicament for the treatment of EBV-infection.
In order to achieve one of the above purposes, the present invention adopts the following technical scheme:
the present invention provides a method for increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations comprising the steps of:
s1, extracting peripheral blood mononuclear cells;
s2, sorting the peripheral blood mononuclear cells to obtain lymphocytes with different subgroup types;
s3, respectively re-suspending lymphocytes of different sub-groups obtained in S2 by using a culture medium, uniformly spreading the re-suspended lymphocytes in a cell culture pore plate, respectively stimulating lymphocytes of different sub-groups spread in the cell culture pore plate by using IL-2 to proliferate the lymphocytes, wherein the final concentration of the IL-2 is 2.9x10 6 ～3.1×10 6 U, after 4-6 h stimulation, collecting the lymphocytes of different sub-populations after proliferation;
s4, respectively extracting the DNA of the lymphocytes of different sub-groups after proliferation in the S3, and measuring the content of the EB virus DNA in the lymphocytes of different sub-groups after proliferation by using a real-time fluorescence quantitative PCR technology.
Further, in the step S2, in the sorting process based on the flow cytometer, a fluorescent-labeled anti-CD3 antibody, a fluorescent-labeled anti-CD19 antibody, and a fluorescent-labeled anti-CD56 antibody are added to the peripheral blood mononuclear cells, and then incubated in a dark place.
Further, the fluorescent-labeled anti-CD3 antibody is anti-CD3-FITC, the fluorescent-labeled anti-CD19 antibody is anti-CD19-PE, and the fluorescent-labeled anti-CD56 antibody is anti-CD56-ECD.
Further, in the step S2, in the sorting process based on the flow cytometer, a fluorescent-labeled anti-CD3 antibody, a fluorescent-labeled anti-CD19 antibody, a fluorescent-labeled anti-CD56 antibody, and a fluorescent-labeled anti-CD45 antibody are added to the peripheral blood mononuclear cells, and then incubated in a dark place.
Further, the fluorescent-labeled anti-CD45 antibody is anti-CD45-PC7.
Further, in the S2, the lymphocytes of the different subpopulations are cd3+ T lymphocytes, cd19+ B lymphocytes and cd56+ NK lymphocytes.
Further, in S2, sorting the peripheral blood mononuclear cells includes magnetic bead-based sorting and/or flow cytometry-based sorting.
Further, in the step S2, the incubation time in the dark is 5-15 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method for improving the sensitivity of identifying the EBV infected lymphocyte subpopulations provided by the invention can quickly and conveniently separate lymphocytes of different subpopulations by using a flow cytometer, stimulate the separated lymphocytes of different subpopulations in vitro for 4-6 hours by using IL-2, stimulate lymphocyte expansion, and improve the sensitivity of measuring the content of EB virus DNA in the stimulated lymphocytes of different subpopulations by using a real-time fluorescent quantitative PCR technology, thereby improving the sensitivity of identifying the EB virus infected lymphocyte subpopulations.
(2) The method for improving the sensitivity of identifying the EBV-infected lymphocyte subpopulation provided by the invention can help to determine the treatment target point of the EBV-infected disease and further guide the development of preparing medicines for treating the EBV infection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following description will briefly introduce the drawings that are needed in the embodiments or the description of the prior art, it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of the total nucleated cells in the sample encircled by the forward and lateral angle parameters provided in example 1.
FIG. 2 is a schematic representation of CD45+ SSC cells, a common phenotype for circle selected lymphocytes, provided in example 1.
FIG. 3 is a schematic representation of a cell population of the general phenotype CD3+CD56-for the selected T cells provided in example 1.
FIG. 4 is a schematic representation of a population of cells with the common phenotype CD3-CD19+ for the encircled B cells provided in example 1.
FIG. 5 is a schematic representation of a cell population of the common phenotype of the encircled NK lymphocytes of example 1 is CD 3-CD56+.
FIG. 6 is a schematic representation of purity detection of CD3+ T lymphocytes using a flow sorter as provided in example 1.
FIG. 7 is a schematic representation of purity detection of CD19+B lymphocytes using a flow sorter as provided in example 1.
Fig. 8 is a schematic diagram of purity detection of cd56+ NK lymphocytes using a flow sorter as provided in example 1, respectively.
FIG. 9 is a schematic diagram showing an amplification curve and a standard curve of an EB virus quantitative standard.
FIG. 10 is a schematic diagram of amplification curves obtained by stimulating CD3+ T lymphocytes, CD19+ B lymphocytes, CD56+ NK lymphocytes after a single patient activation with IL-2 and then performing DNA detection of EBV, respectively, and EBV DNA copy numbers calculated from the standard curve in example 1.
FIG. 11 is a schematic representation of the amplification curve obtained by stimulating CD3+ T lymphocytes, CD19+ B lymphocytes, CD56+ NK lymphocytes after a single patient activation without IL-2 followed by EBV DNA detection and the EBV DNA copy number calculated from the standard curve in example 1.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the technical schemes of the present application will be clearly and completely described below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.
Example 1
(1) Collecting peripheral blood of clinical patient
Clinical EBV DNA copy number of not less than 10 in peripheral blood 3 copy/mL patient, sign informed consent, and collect 3mL peripheral blood using EDTA tube vein after detection and patient status assessment prior to conventional treatment.
(2) Flow cytometry sorting lymphocytes
S1, lysing red blood cells: transferring 1mL of peripheral blood into a 15mL centrifuge tube, adding 14mL of 8.3 mass percent ammonium chloride solution, fully and uniformly mixing, standing for more than 15min to fully crack red blood cells, centrifuging the sample at the centrifuge speed of 1000rpm and the temperature of 4 ℃ for 10min to precipitate white blood cells in the sample at the bottom of the centrifuge tube, and after centrifuging, discarding supernatant liquid of the centrifuged sample to preserve white blood cell precipitation at the bottom of the sample to about 100uL;
s2, labeling lymphocytes by using a fluorescent-labeled antibody: respectively adding 10uL of anti-CD3-FITC antibody, 10uL of anti-CD19-PE antibody, 10uL of anti-CD56-ECD antibody and 5uL of anti-CD45-PC7 antibody into the leukocyte pellet, and incubating for 5-15 min in a dark place;
s3, separating lymphocytes of different sub-group types by using a flow cytometer: the fluorescence-labeled antibody-labeled lymphocyte samples were detected and sorted using a Beckman Coulter MoFlo flow sorter (Beckman, USA), as follows:
1. all nucleated cells in the sample to be detected are selected in circles through the parameters of the forward angle and the lateral angle, are set to be nucleated cell gates, and cell fragments in the sample are removed, as shown in the figure 1;
2. a population of lymphocytes, a common phenotype, cd45+ SSC cells, was selected from the phylum nucleated cells by anti-CD45-PC7 antibody markers and lateral angle parameters, and set up as phylum lymphocytes to select lymphocytes, as shown in figure 2;
3. the cell population with the common phenotype of CD3+CD56-of T cells is circled from lymphocyte gate through anti-CD3-FITC antibody label and anti-CD56-ECD antibody label, and is set as the T cell gate for sorting, as shown in figure 3;
4. cell populations with the common phenotype of CD3-CD19+ for B cells were selected from the lymphophylum by circling the anti-CD3-FITC antibody label with the anti-CD19-PE antibody label, and were set to the B cell phylum for sorting, as shown in FIG. 4;
5. the cell population with the common phenotype of NK lymphocytes of CD3-CD56+ is circled from the lymphocyte gate by the anti-CD3-FITC antibody label and the anti-CD56-ECD antibody label and is set as the NK lymphocyte gate for sorting, as shown in figure 5;
6. after sorting T, B, NK lymphocyte populations and gating, sorting logic is set in the flow sorter, which is a single cell-purity mode (purity), i.e., when a droplet contains one and only one cell that fully meets a predetermined fluorescent label, the droplet will be sorted into a collection container;
7. preparing corresponding flow pipes and marking, adding 100uL of 0.9 mass percent normal saline into each flow pipe, then debugging a flow sorter to sort liquid flow, and sorting according to preset logic after preparation;
8. after the start of the sorting, the number of cells obtained at each cell gate of T, B, NK was recorded by a flow sorter, and when the number of cells at each cell gate of T, B, NK reached 2X 10 5 Stopping sorting and recording data, so as to sort and obtain CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes from the sample to be tested;
s4, respectively detecting the purity of the separated CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes by using a flow cytometer: after the sorting is finished, respectively centrifuging the liquid with CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes obtained by sorting at the rotation speed of 1000rpm for 10min at 4 ℃ for centrifugation, and discarding the upper liquid after the cells are precipitated to the bottom; and a proper amount of samples are sucked, purity of the CD3+ T lymphocytes, CD19+ B lymphocytes and CD56+ NK lymphocytes obtained by sorting is detected by a Beckman Navios flow meter, when the purity of the CD3+ T lymphocytes, CD19+ B lymphocytes and CD56+ NK lymphocytes is detected to be more than 85%, the sorting result is ideal, the obtained cells are used for subsequent experiments, and as can be seen from figures 6-8, the purity of the CD3+ T lymphocytes, CD19+ B lymphocytes and CD56+ NK lymphocytes obtained by sorting is more than 95%, and the cells are qualified;
it should be noted that the above lymphocyte sorting method can also be performed based on magnetic beads, and this is not described herein because the technology is mature.
S5, after sorting is completed, stimulating lymphocyte proliferation by using IL-2:
1. respectively re-suspending the sorted CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes by using 1mL of culture medium, and uniformly spreading the re-suspended CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes in a 24-well plate;
2. adding IL-2 into the culture holes of CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes in a 24-hole plate respectively to ensure that the cells continue to proliferate, wherein the final concentration of the IL-2 is 2.9X106-3.1X106U, and collecting the proliferated CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes after 4-6 h stimulation;
s6, detecting DNA of EBV in CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes by real-time fluorescence quantitative PCR:
1. transferring the cell suspensions of the proliferated CD3+T lymphocytes, CD19+B lymphocytes and CD56+NK lymphocytes into a 1.5mL centrifuge tube respectively, centrifuging for 1min at the room temperature at the speed of 12000rpm, and carefully sucking and discarding the supernatant to keep the sediment after centrifuging;
2. vortex vibration mixing nucleic acid extract, sucking 50uL nucleic acid extract, adding into 1.5mL centrifuge tube containing cell sediment, vortex vibration mixing, dispersing sediment, and maintaining at 100deg.C for 10min;
3. respectively placing 1.5mL centrifuge tubes on a centrifuge, and centrifuging for 2min at the room temperature of 12000 rpm;
4. centrifuging, wherein supernatant of the centrifuge tube is respectively DNA of CD3+T lymphocytes, DNA of CD19+B lymphocytes and DNA of CD56+NK lymphocytes, and the supernatant is absorbed for PCR detection or preservation at-20 ℃;
5. the PCR reaction liquid is prepared according to the following system: PCR buffer solution X9 uL, taq enzyme X3 uL, EB virus primer probe X8 uL, 20uL of each tube are split-packed into PCR reaction tubes;
6. according to EB virus quantitative standard S1-S4 (EB virus plasmid E.coli, S1: 5X 10) 7 copy/mL, S2: 5X 10 6 copy/mL, S3: 5X 10 5 copy/mL, S4: 5X 10 4 copy/mL), strong positive control (EB virus plasmid E.coli, 1.7X10) 5 -1.7×10 6 copy/mL), critical positive control (EB virus plasmid E.coli, 1.3X10) 3 -1.4×10 4 copy/mL), negative control (negative plasma), and the sequence of the sample to be tested, respectively taking 10uL of DNA respectively, adding into a PCR reaction tube, centrifuging at a low speed for several seconds, and placing on a real-time fluorescence quantitative PCR instrument;
7. PCR amplification procedure: reacting at 50 ℃ for 2min, preserving heat at 94 ℃ for 5min, and circulating for 1 time; cycling for 5 times at 94 ℃,10s,60 ℃,45 s; FAM and ROX channel signals are collected at the temperature of 60 ℃ after being cycled for 40 times at 94 ℃,10s,60 ℃ and 45 s.
8. Constructing a standard curve according to an EB virus quantitative standard, and then respectively calculating the detection sensitivity of EBV DNA in the proliferated CD3+T lymphocytes, the detection sensitivity of EBV DNA in the CD19+B lymphocytes and the detection sensitivity of EBV DNA in the CD56+NK lymphocytes in the sample to be detected according to the standard curve, wherein the amplification curve and the standard curve of the EB virus quantitative standard are shown in the figure 9; the amplification curve of EBV DNA detection and EBV DNA copy number calculated from the standard curve after sorting T lymphocytes, B lymphocytes and NK lymphocytes of an EBV positive patient and stimulating with IL-2 are shown in FIG. 10; the amplification curve of EBV DNA detection and the EBV DNA copy number calculated from the standard curve without IL-2 stimulation after sorting of T lymphocytes, B lymphocytes, NK lymphocytes of one EBV positive patient are shown in FIG. 11.
As can be seen from FIG. 11, the EBV positive patients detected EBV-positive DNA in CD19+B lymphocytes, CD3+T lymphocytes, and CD56+NK lymphocytes without IL-2 stimulation of the sorted CD3+T lymphocytes, CD19+B lymphocytes, CD56+NK lymphocytes, and then EBV-positive DNA detectionThe Cp value of the EBV DNA in the DNA and CD19+B lymphocytes is 30.63, and the sensitivity of the real-time fluorescence quantitative PCR detection of the EBV DNA in the CD19+B lymphocytes in a sample to be detected is 1.36 multiplied by 10 3 copy/mL; cp value of EBV DNA in CD3+T lymphocyte is 32.22, and sensitivity of real-time fluorescence quantitative PCR detection of EBV DNA in CD3+T lymphocyte in sample to be detected is 2.82×10 2 copy/mL, indicating EBV infection of B lymphocytes and NK lymphocytes in lymphocyte subpopulations in this case.
As can be seen from FIG. 10, the use of IL-2 to stimulate the sorted CD3+ T lymphocytes, CD19+ B lymphocytes and CD56+ NK lymphocytes can continue to proliferate the sorted CD3+ T lymphocytes, CD19+ B lymphocytes and CD56+ NK lymphocytes, and then the EBV DNA detection is performed respectively, which shows that the EBV positive patient only detects the EBV DNA in the CD19+ B lymphocytes and CD3+ T lymphocytes, but the CD56+ NK lymphocytes do not detect the EBV DNA, the Cp value of the EBV DNA in the CD19+ B lymphocytes is 29.51, and the sensitivity of the real-time fluorescence quantitative PCR detection of the EBV DNA in the CD19+ B lymphocytes in the sample to be tested stimulated by IL-2 is 1.32X10 3 copy/mL; cp value of EBV DNA in CD3+ T lymphocytes 28.95, and sensitivity of real-time fluorescence quantitative PCR detection of EBV DNA in CD3+ T lymphocytes in a sample to be tested stimulated with IL-2 was 1.94X10 2 copy/mL, demonstrating that the sensitivity of EBV-infected lymphocyte subpopulations in this example was identified using the method of the invention provided for increasing the sensitivity of EBV-infected lymphocyte subpopulations in patients, and that the sensitivity of EBV DNA calculated from the standard curve in CD19+ B lymphocytes, CD3+ T lymphocytes in this example using IL-2 stimulation was higher than that of EBV DNA in CD19+ B lymphocytes, CD3+ T lymphocytes without IL-2 stimulation, as compared to the results of DNA detection of EBV using IL-2-stimulated sorted lymphocytes and IL-2-stimulated sorted lymphocytes, it can be seen that the Cp value of EBV DNA obtained using IL-2-stimulated sorted CD3+ T lymphocytes, CD19+ B lymphocytes, CD56+ NK lymphocytes was lower than that of EBV DNA in EBV-infected subpopulations without IL-2 stimulation, and that the sensitivity of EBV DNA calculated from the standard curve in EBV-infected cell subpopulations in this example using IL-2 stimulation was higher than that of EBV-infected cell subpopulations using the invention provided for increasing the sensitivity of EBV-infected cell subpopulationsThe method can improve the sensitivity of real-time fluorescence quantitative PCR technology for measuring the content of EB virus DNA in lymphocytes of different subgroup types, thereby improving the sensitivity of identifying the lymphocyte subgroup infected by the EB virus. The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (10)
1. A method for increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations, characterized in that,
the method comprises the following steps:
s1, extracting peripheral blood mononuclear cells;
s2, sorting the peripheral blood mononuclear cells to obtain lymphocytes with different subgroup types;
s3, respectively re-suspending lymphocytes of different sub-groups obtained in S2 by using a culture medium, uniformly spreading the re-suspended lymphocytes in a cell culture pore plate, respectively stimulating lymphocytes of different sub-groups spread in the cell culture pore plate by using IL-2 to proliferate the lymphocytes, wherein the final concentration of the IL-2 is 2.9x10 6 ～3.1×10 6 U, after 4-6 h stimulation, collecting the lymphocytes of different sub-populations after proliferation;
s4, respectively extracting the DNA of the lymphocytes of different sub-groups after proliferation in the S3, and measuring the content of the EB virus DNA in the lymphocytes of different sub-groups after proliferation by using a real-time fluorescence quantitative PCR technology.
2. The method according to claim 1, wherein in S2, the fluorescent-labeled anti-CD3 antibody, the fluorescent-labeled anti-CD19 antibody and the fluorescent-labeled anti-CD56 antibody are added to the peripheral blood mononuclear cells during the sorting by the flow cytometer, and then incubated in the dark.
3. The method of claim 2, wherein the fluorescently labeled anti-CD3 antibody is anti-CD3-FITC, the fluorescently labeled anti-CD19 antibody is anti-CD19-PE, and the fluorescently labeled anti-CD56 antibody is anti-CD56-ECD.
4. The method according to claim 1, wherein in S2, the fluorescent-labeled anti-CD3 antibody, the fluorescent-labeled anti-CD19 antibody, the fluorescent-labeled anti-CD56 antibody and the fluorescent-labeled anti-CD45 antibody are added to the peripheral blood mononuclear cells during the sorting by the flow cytometer, and then incubated in a dark place.
5. The method of claim 4, wherein the fluorescent-labeled anti-CD45 antibody is anti-CD45-PC7.
6. A method of increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations according to claim 1, wherein in said S2, said different subpopulations of lymphocytes comprise cd3+ T lymphocytes, cd19+ B lymphocytes and cd56+ NK lymphocytes.
7. The method of claim 1, wherein in S2, sorting the peripheral blood mononuclear cells comprises magnetic bead-based sorting and/or flow cytometry-based sorting.
8. A method of increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations according to any one of claims 2 or 4, wherein said incubation in S2 is performed for a period of 5 to 15 hours in the absence of light.
9. Use of a method according to any one of claims 1 to 8 for increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations in determining a therapeutic target for EBV-infectious disease.
10. Use of a method according to any one of claims 1 to 8 for increasing the sensitivity of identifying EBV-infected lymphocyte subpopulations for guiding the preparation of a medicament for the treatment of EBV infection.
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